Mesenchymal stem cells (MSCs) pose exciting possibilities for cell-based regenerative therapies. Their capability to differentiate along multipe musculoskeletal lineages makes them attractive as an alternative source of autologous cells. However, these cells must first be separated from other cell types before practical use, which has proven difficult using antibody-based sorting approaches. Typical cell yields are often less than 1%, which necessitates expensive and time-consuming monolayer expansion to obtain sufficient cell numbers for clinical therapies. Alternative enrichment strategies are needed that can identify large numbers of cells by lineage potential. In this project, we will investigate mechanical- and gene expression-based approaches for osteogenic and adipogenic lineage enrichment. To accomplish the overall goals of the project we will: (1) Determine whether live, single-cell mechanical biomarkers indicate the regenerative capacity of stem cells. Atomic force microscopy will be used to quantify the elastic and viscoelastic properties of individual MSCs before, during, and after osteogenic and adipogenic differentiation. These data will provide sorting parameters that allow for separation of cells into tissue-specific groups based on mechanical similarity. The tissue building capacity of sorted cell populations will be compared using established in vitro protocols for osteogenesis and adipogenesis. (2) Develop and implement a live-cell molecular beacon assay that distinguishes differentiating cells from non-differentiating cells based on lineage- specific gene expressions. Beacons, which fluoresce upon binding to target mRNA molecules, will identify osteogenic and adipogenic potential in freshly isolated MSCs from lipoaspirate. Highly expressing cells will be enriched via fluorescence-activated cell sorting and evaluated for their differentiation capabilities. (3) Investigate the combination of mechanical and gene expression biomarkers for enrichment of lineage-specific cells in vitro and in vivo. Preliminary findings suggest that molecular beacons can identify a broad set of cells capable of lineage-specific differentiation while mechanical biomarkers can predict the robustness of the differentiation response. The combination of these two techniques is expected to enrich for highly regenerative, tissue-specific cells. Sorted cell populations will be evaluated for the amount and type of matrix synthesized, first, in monolayer and, ultimately, in an in vivo mouse model. In addition to discovering how mechanical properties are associated with differentiation and lineage potential, this work will generate a set of molecular beacons broadly applicable to musculoskeletal investigations. We expect experimental findings to elucidate aspects of cellular heterogeneity in stem cell populations while also facilitating the practical implementation of cell-based clinical therapies. This work wil be conducted through collaborations between Brown University and Rhode Island Hospital.